Antibody against a mast cell surface antigen

ABSTRACT

A mast cell surface antigen, DNA thereof and an antibody against the antigen are provided. The amino acid sequence of this mast cell surface antigen is the translation of the coding region of its DNA. The base sequence of this DNA has been clarified in the following manner. Namely, mast cells obtained by incubating cord blood monocular cells are co-incubated with primary culture of fibroblasts to give connective tissue type mast cells (MC-TC). Then mRNA is extracted from this MC-TC cell extraction and a cDNA library is constructed therefrom. Immunological screening is carried out with the use of anti-MC-TC antiserum and the base sequence of the positive clone thus obtained is identified. Owing to the clarification of the amino acid sequence of this mast cell antigen, it becomes possible to reveal the role of mast cells in the pathology of allergic diseases and thus an antibody against mast cells can be easily obtained.

This application is a divisional application of Ser. No. 10/250,644filed Jan. 4, 2001, now U.S. Pat. No. 7,045,597, which is a nationalstage completion of PCT/JP01/00005 filed Jul. 3, 2001 which is acontinuation-in-part of Ser. No. 09/229,932 filed Jan. 13, 1999, nowU.S. Pat. No. 6,255,107.

TECHNICAL FIELD OF THE INVENTION

This invention relates to a mast cell surface antigen, DNA thereof, andantibody against the antigen.

BACKGROUND OF THE INVENTION

Allergic diseases, such as bronchial asthma or allergic rhinitis, areinduced as follows: firstly, antigen-specific IgE production is induced,and then, from mast cells, basophils and the like, activated by theinduced IgE, various chemical mediators such as histamine, eosinophilchemotactic factor in allergy (ECF-A), leukotrienes, platelet-activatingfactor (PAF) and thromboxane are produced and released. Specifically intissues, the mast cells release these chemical mediators, and thereforeplay an important role in development of allergic diseases.

Human mast cells are differentiated into tryptase positive cell (MC-T)and both tryptase and chymase positive cells (MC-TC), according to thegranule content of proteolytic enzyme in the mast cells. MC-T are mainlydistributed in the lung tissues and the gastrointestinal tract mucosa,whereas MC-TC are distributed in the skin tissues. These mast cells,unlike cells of other leukocytes, leave bone marrow for the peripheralenvironment as pluripotent stem cells, and differentiate into MC-T orMC-TC, followed by adhesion to either lung or skin fibroblasts. Since itis believed that such mast cells play a major role in development ofallergic diseases, it is necessary to specifically detect and separatethe mast cells in order to clarify the physiological functions of themast cells.

However, heretofore, no cell surface antigen specific to the mast cellshas been known. Since the antibody against the cell surface antigenspecific to the mast cells will allow us to specifically remove oreliminate the mast cells, identification of the cell surface antigenspecific to the mast cells has an important meaning not only inclarification of the underlying cause of allergic diseases but intreatment thereof.

An object of the present invention, which was made to solve the aboveproblem, is to provide a mast cell surface antigen, DNA thereof, and anantibody against the antigen.

DISCLOSURE OF THE INVENTION

The mast cell surface antigen comprises an amino acid sequence listed inSEQ ID NO. 1, or a substantially identical amino acid sequence. Thisamino acid sequence is a translation of the coding region of DNA of themast cell surface antigen. The base sequence of the DNA of the mast cellsurface antigen has been clarified in the following manner. Namely, asexplained in detail in an embodiment section, after mast cells areobtained from cord blood monocular cells, mRNA is extracted from cellextraction of these mast cells, and a cDNA library is constructed fromthe mRNA. Immunological screening of the cDNA library is carried outusing the antiserum, and the base sequence of the positive clone thusobtained is identified by means of a DNA sequencer. In the base sequencelisted in SEQ ID NO. 2, a sequence of 36-38, namely ATG, is theinitiation codon, and a sequence of 2394-2396, namely, TGA, is thetermination codon. In other words, a sequence of 36-2396 is the codingregion, and the base sequence in the range codes the amino acid sequencelisted in SEQ ID NO. 1. Identification of the amino acid sequence ofthis mast cell antigen allows us to clarify the role of the mast cellsin development of allergic diseases, and thus to obtain the antigenwhich specifically reacts to the mast cells.

An antibody against the mast cell surface antigen can be obtained in thefollowing steps, for example. Firstly, the mast cell antigen comprisingthe amino acid sequence listed in SEQ ID NO. 1 is injected to a mammal(except human) for immunization, and fused cells are prepared by fusingthe antibody producing cells obtained from the immunized mammal withmyeloma cells. Then, from the fused cells, a clone which produces anantibody that reacts with the mast cell surface antigen is selected andcultured, and the supernatant of the culture is purified. The antibodyallows us to specifically remove or eliminate the mast cells, and thusto treat allergic diseases. In short, this antibody is expected to workas antiallergic agent.

Cells that produce this antibody can be obtained in the following steps,for example. Firstly, the mast cell antigen comprising the amino acidsequence listed in SEQ ID NO. 1 is injected to a mammal (except human)for immunization, and fused cells are prepared by fusing the antibodyproducing cells obtained from the immunized mammal with myeloma cells.Then, from the fused cells, a clone which produces an antibody thatreacts with the mast cell surface antigen is selectively cultured.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the ratio of chymase positive mast cells andmeasured values of tryptase concentration for the mast cells before andafter coculture;

FIG. 2 is an explanatory view showing base Bcsc-1; and

FIG. 3 is an explanatory view showing a construction of a cDNAexpression vector BCMGSNeo.

BEST MODE FOR CARRYING OUT THE INVENTION

[1] Culture of MC-TC

Cord blood treated with heparin was layered over the Ficoll Hypaquesolution (specific gravity: 1.077, Sigma Inc.), centrifuged at 300×g for30 minutes at room temperature to separate the mononuclear cells, whichwere then suspended in RPMI1640 medium (Nissui Seiyaku) containing 10%of FBS (Gibco BRL), 50ìM of 2-mercaptoethanol, 4 mM of L-glutamine, 100U/ml of penicillin and 50ìg/ml of streptomycin. The concentration of themononuclear cells in the suspension was adjusted to 5×10⁶/ml, and thesuspension was poured into a collagen coated culture dish (Iwaki Glass)having a diameter of 10 cm, and, added with SCF (100 ng/ml, PeproTechInc.) and IL-6 (50 ng/ml, PeproTech Inc.), cultured for 2 weeks toobtain two-week cultured cells containing neutrophils, lymphocytes,macrophages, basophils and precursor cells of mast cell. SCF is a factorparticipating in differentiation and proliferation of mast cellsexpressed on fibroblasts. It is an abbreviation for stem cell factor.

The cord blood mononuclear cells obtained in the above were cultured inthe presence of 100 ng/ml of SCF and 50 ng/ml of IL-6 for 6 weeks, andwhen the human mast cells became predominant, that is, when the numberof human mast cells reached the order of 106, the mast cells werefurther cocultured with a primary culture of human fibroblasts.Specifically, the human mast cells were transferred to a monolayer ofhuman fibroblasts from either skin or lung tissues, and cultured for 2months in the presence of 50 ng/ml SCF.

The ratio of chymase positive mast cells and the concentration oftryptase were measured for the mast cells before and after thecoculture. The result is shown in FIG. 1. FIG. 1 shows that before thecoculture of mast cells, i.e. for the human mast cells cultured for10-16 weeks in the presence of SCF and IL-6 (the left end of the graphin FIG. 1), the ratio of chymase positive cells and the concentration oftryptase were both very low, whereas those for the human mast cellsafter cocultured with human fibroblasts for 6-8 weeks (the center of thegraph in FIG. 1) and those for the human mast cells cocultured with lungfibroblasts (the right end of the graph in FIG. 1) showed remarkableincreases, especially for the human mast cells cocultured with skinfibroblasts.

Staining was performed to the mast cells cultured for 15 weeks in thepresence of SCF and IL-6 and the mast cells cocultured with skinfibroblasts for 2 months after having been cultured for 6 weeks, usingantibodies against tryptase and those against chymase. As a result, incase of staining against tryptase, it was confirmed that both types ofmast cells were wholly stained, whereas in case of staining againstchymase, it was confirmed that most cells cocultured with fibroblastwere stained but cells which were not cocultured were only partlystained. The results show that MC-T is differentiated into MC-TC by thecoculture.

As described above, connective tissue type-human mast cells, or MC-TC,were obtained by culturing cord blood monocular cells in the presence ofSCF and IL-6 and subsequently coculturing them with the primary cultureof human skin fibroblasts.

[2] Preparation of mRNA

According to the manual of “Quick-Prep Micro mRNA Purification Kit” ofPharmacia, poly(A)+mRNA was extracted from the cell extraction of theabove MC-TC in the following steps.

2×10⁶ of MC-TC was centrifuged and washed with PBS twice, added with400ìL of elution buffer accompanying the aforementioned kit, stirredwell by a vortex mixer (rotary beater), further added with 800ìL ofelution buffer and stirred by the vortex mixer. The solution obtainedwas transferred into an assist tube (Assist) made of polypropyrene, andcentrifuged at 14,000 rpm for 5 minutes to separate the precipitate.

1 mL of oligo dT cellulose was centrifuged at 14,000 rpm to separate thesupernatant.

The oligo dT cellulose in 2) was added to the cell extraction in 1), andthe solution was well mixed by inverting the assist tube for 3 minutes,centrifuged at 14,000 rpm for 5 seconds to separate the supernatant,which was then washed with 1 mL of high salt concentration buffer 5times and then in low salt concentration buffer 2 times.

The precipitate was suspended in 300ìL of low salt concentration buffer,put in a microspin column accompanying the aforementioned kit, which wascentrifuged for 5 seconds, and washed with 500ìL of low saltconcentration buffer 3 times.

200ìL of elution buffer warmed up to 65° C. was added to the column, and10ìL of glycogen solution and 400ìL of potassium acetate solution werefurther added. After 1 mL of 95% ethanol was added, the column wascentrifuged at 14,000 rpm for 1 hour at 4° C., and dried under reducedpressure.

[3] Construction of cDNA Library

According to the manual of “Zap-cDNA synthesis kit” of Stratagene, acDNA library was constructed from mRNA obtained in the above [2]. Sizefractionation was carried out using CL-2B gel accompanying this kit.

[4] Immunological Screening

A phage solution of the cDNA library constructed in the above [3] wasdiluted with SM buffer (containing 50 mM of Tris-HCl (pH7.5), 100 mM ofNaCl, 10 mM of MgSO₄.H₂O, and 0.01% gelatin), to give a phageconcentration of 2.5-4×10⁵ pfu/mL. This solution was dispensed per 100ìLto 10 centrifuging tubes. Added with 500ìL of overnight culture solutionof Escherichia coli (XL-1blue) (which was cultured overnight in 5 mL ofNZY medium so as to obtain O.D.₆₀₀=1.5), the solution was stirred with avortex mixer, and incubated at 37° C. for 15 minutes. Further added with7 mL of soft agarose (which was made to give a concentration of 0.6%, bydissolving agarose gel for electrophoretic migration in a culturemedium), the solution was stirred by the vortex mixer, and spread in a14×10 cm square dish. When the agarose was set, the dish was soaked in20 mL of IPTG, and with a dried nitrocellulose filter thereon, incubatedat 37° C. for 4 hours. For the NZY medium, 5 g of NaCl, 2 g ofMgSO₄.7H₂O, 5 g of yeast extract, and 5 g of NZamine (caseinhydrolysate) were dissolved in 1 liter of water, the pH was adjusted to7.5 with NaOH, the solution added with 15 g agar was sterilized anddissolved in an autoclave.

After the incubation, the filter was removed, and washed with shakingwith 300 mL PBS for 15 minutes, which was repeated 2 times. Then, thePBS was discarded. Added with 300 mL of PBS (PBS-SM) containing 0.5%skim milk, the filter was washed with shaking for 1 hour at roomtemperature for blocking. Added with a rat antiserum (which is anantiserum obtained from a rat which provided its spleen cells forpreparation of a later-described hybridoma clone ahMC5C12 (Accession No.FERM BP-6070)) diluted 1:400 in PBS-SM, the filter was washed withshaking for 1 hour at room temperature. The antiserum solution wasdiscarded, and the solution was washed with shaking with PBS plus 0.05%Tween (PBS-T) for 5 minutes at room temperature, which was repeated 4times. Then, added with 200 mL of peroxydase labeled anti-rat IgG(Medical & Biological Laboratories Co., Ltd.) diluted 1:1000 in PBS-SM,the filter was washed with shaking for 1 hour at room temperature,washed with shaking 4 times with 500 mL of PBS-T, and further twice with100 mL of PBS.

The filter was drained, soaked in a color substrate solution (solutionof 12 mg of diaminobenzidine/25 mL of PBS, added with 50ìL of 2.5%cobalt chloride and 50ìL of 2% nickel sulphate), and further added with80ìL of 30% hydrogen peroxide solution for color development. Based onthe location of color development, associated clones were selected.

[5] Determination of Base Sequence

According to the manual of the aforementioned “Zap-cDNA synthesis kit”of Stratagene, in vivo excision of 2 positive clones obtained byimmunological screening from the Uni-ZAP XR vector was carried out. Theclones were subcloned in pBluescript phagemid and grown with Escherichiacoli. The base sequences of the respective clones were determined usingABI PRISM 377 DNA sequencer of Perkin Elmer.

In homology search by BLAST conducted against the database for theobtained base sequences, the base sequence of one clone wasapproximately coincided with that of tryptase III, and the base sequenceof the other clone was approximately coincided with that of a gene namedBreast cancer suppressor candidate-1 (Bcsc-1) (FIG. 2). In FIG. 2, SEQID NO. 8, a sequence of 1-2552 represents Bcsc-1. BLAST is anabbreviation of Basic Local Alignment Search Tool.

A clone having the approximately identical base sequence to that oftryptase III was determined to have nothing to do with the target cellsurface antigen, since tryptase III is a well-known enzyme which existsin mast cell granules. Then, it was determined whether the clone havingthe approximately identical base sequence to that of Bcsc-1 is thetarget cell surface protein.

[6] Transfection to BHK Cells

Among the cDNA obtained in the above, the part designated as a regionfor coding protein of Bcsc-1 stored in the database was inserted to anexpression vector which was prepared according to the method ofKarasuyama et al. (see FIG. 3; Karasuyama, H. & Melchers, F.: Eur. J.Immunol., 18, 97-104, 1988, Karasuyama, H., Tohyama, N. & Tada, T.: J.Exp. Med., 169, 13-35, 1989, Yagita, H., Nakamura, T., Karasuyama, H. &Okumura, K.,: Proc. Natl. Acad. Sci. USA, 86, 645-649, 1989, Karasuyama,H., Kudo, A. & Melchers, F.: J. Exp. Med., 172, 969-972, 1990).Particularly, in introduction of XhoI and NotI restriction enzymefragments into Bcsc-1, cagCTCGAGatggaggaggctctgggg (SEQ ID NO. 3) wasused as 5′ XhoI primer, and tctggatGCGGCCGCtcaaaggcaaagat (SEQ ID NO. 4)was used as 3′ NotI primer. As PCR conditions, the Bcsc-1 was heated at94° C. for 4 minutes, reacted at 95° C. for 1 minute and 15 seconds, at60° C. for 1 minute and 10 seconds, and 72° C. for 3 minutes, which wasrepeated 39 times, and then reacted at 72° C. for 5 minutes. The Bcsc-1introduced with XhoI and NotI site was treated with XhoI and NotI, andthen inserted to BCMGSNeo which was also treated with XhoI and NotI.

The BHK cells were introduced to Bcsc-1 by lipofection and geneticallytransformed. Namely, the BHK cells were spread in 10% FCS DMEM (NissuiSeiyaku) to give a density of 50-70%, and after cultured overnight,cultured at 37° C. for 2 hours in a fresh medium. The BHK cells werewashed 3 times with serum-free DMEM. 100ìL of serum-free DMEM containing2.5 ìL (2.5 ìg) of Bcsc-1 introduced BCMGSNeo, and 200ìL of equivalentmixture of 100ìL of serum-free DMEM containing 100ìL of lipofectaminwere added to 800ìL of serum-free DMEM, and the BHK cells were culturedat 37° C. for 3 hours.

[7] Preparation of anti-Bcsc-1 Antibody

Bcsc-1 introduced pET-28a vector was expressed in Escherichia coli as afusion protein with His-tag. The fusion protein was purified on a nickelchelate column, and a rabbit was immunized with the purified protein ina usual manner, to prepare an antiserum, namely, anti-Bcsc-1 polyclonalantibody. Introduction of Bcsc-1 to pET-28a vector was carried out asfollows. EcoRI and XhoI restriction fragments were introduced to Bcsc-1,and after treated with XhoI and EcoRI, the Bcsc-1 was inserted topET-28a vector which was also treated with XhoI and EcoRI. Theintroduction of EcoRI and XhoI restriction fragments to Bcsc-1 werecarried out by PCR amplification using tcagGAATTCatggaggaggctct (SEQ IDNo. 5) as 5′ EcoRI primer and ggtaCTCGAGaaaggcaaagatagc (SEQ ID No. 6)as 3′ XhoI primer.

[8] Cell Staining and Western Blot

The BHK cells obtained in the above [6] (which were expressed byinsertion of Bcsc-1 gene) were cultured overnight in 10% FCS DMEMcontaining 500ìg/mL of G418 to separate the culture. The anti-Bcsc-1polyclonal antibody (diluted 1:10³) obtained in the above [7] was placedon the culture, which was then reacted at 37° C. for 1 hour, and rinsedwith PBS. Further added with FITC labeled anti-rabbit IgG (H+ L)(Medical & Biological Laboratories Co., Ltd.) (diluted 1:100), theculture was reacted at 37° C. for 1 hour to observe localization withinthe cells by a fluorescence microscope.

Also, protein recognized by this anti-Bcsc-1 polyclonal antibody wassearched by Western blot, using mast cell extract, Bcsc-1 introduced BHKcell extract, extract of eosinophil in which differentiation was inducedusing IL-5, peripheral blood cell extract, extract of HL-60, that is, aperipheral blood system cell line, and extract of eosinophildifferentiated from HL-60, as antigens.

As a result, when the mast cell extract was stained, diffusefluorescence was recognized in the whole cytoplasma. Accordingly, itseemed that the Bcsc-1 gene is not against the mast cell surfaceantigen. In Western blot, when the Bcsc-1 introduced BHK cells wereantigens, a band was detected at around 45 kD, which was substantiallycoincided with molecular weight expected from the inserted Bcsc-1. Onthe other hand, when the mast cell was the antigen, a band wasrecognized at around 90 kD. The difference in the results between 2 cellextracts gives support to a fact that the Bcsc-1 gene is not against themast cell surface antigen.

[9] Determination of Base Sequence

The gene sequence found by the present inventors was compared with thatof Bcsc-1 (SEQ ID NO. 8) stored in the database (FIG. 2). As a result,the base sequence obtained by the present inventors had one extra base A(943 in FIG. 2 or 943 of SEQ ID NO. 2) around 140 bp upstream of theinitiation codon (ATG of 1055-1057 in FIG. 2) of Bcsc-1. Accordingly, itbecame apparent that the initiation codon of the base sequence obtainedby the present inventors (36-38 in FIG. 2 or 36-38 of SEQ ID NO. 2) islocated around 1000 bp upstream of the Bcsc-1 initiation codon.Furthermore, the region containing a newly found initiation codon was atypical transcription initiation sequence (ACCATGG). The protein codedby the base sequence obtained by the present inventors (SEQ ID NO. 2)was named MASA-1.

[10] Introduction of MASA-1 to BHK Cells

Transfection of the BHK cells by MASA-1 was carried out in the samemanner as in the above [6]. However, tcttgcCTCGAGatggtgcacttctgtgg (SEQID NO. 7) was used as 5′ XhoI primer.

[11] Determination of MASA-1 Properties

Using the MASA-1 introduced BHK cells instead of the Bcsc-1 introducedBHK cells, cell staining and Western blot were performed in the similarway to the above [8]. However, in cell staining, a monoclonal antibody5C12 produced by a below mentioned hybridoma ahMC5C12 was used as aprimary antibody. As a result, a band at around 90 kD was observed byWestern blot using the MASA-1 introduced BHK cells as an antigen, as inthe case of using the mast cells, and cell surface expression wasobserved as well by cell staining. Expression in RBL-2H3 rat mast cellswas also observed. However, no expression was observed in cord blood,eosinophil, peripheral blood lymphocyte (PBL), and HL-60 peripheralblood cell line. From the above, it was confirmed that the protein codedby MASA-1 is a mast cell surface specific protein.

[12] Hybridoma ahMC5C12 (Accession No. FERM BP-6070)

Here, as an example, a method for preparing the hybridoma ahMC5C12 isexplained. Firstly, the cells obtained from cord blood cells culturedfor 2 weeks in Embodiment 1 (10⁶ cells/0.10 ml) was injected into theabdominal cavity of an infant rat 4 days after birth to ablate theinduction ability of antibody production for all antigens in the cells.Then, 1.5 months later, the rat was injected to its abdominal cavitywith both the MC-TC (10⁶ cells/0.10 ml) obtained in the above [1] andcomplete adjuvant for immunization. Further, the rat was injected to itsabdominal cavity with the cells alone 2 times every 2 weeks. The spleenwas taken out 4 days after the final immunization and the cell fusionwas carried out as follows.

Spleen cells taken out from the rat and myeloma cells of a mouse weremixed in a ratio of 10:1, and added with 50% polyethylene glycol 1500 asa fusion accelerator to carry out cell fusion. After the cell fusion,the cells were suspended in HAT medium containing 10% bovine serum insuch a way that the cell concentration for a spleen cell becomes 5□˜10⁵cells/ml, and distributed to a 96 well microtiter plate (Nalge NuncInternational) by 200ìl for every well. The hybridomas were cultured ina CO₂ incubator (5% CO₂, 37□{hacek over (Z)}), and grown in HAT medium.Screening of the hybridomas of spleen cells and myeloma cells wascarried out. The cells were then adapted and cultured in IMDM (Iscove'smodified Dulbecco's medium) supplemented with 10% FCS (Fetal CalfSerum). Among antibodies in the culture supernatant of the hybridomas,by using MC-TC separated from skin lesion of mastocytoma as antigens,clones which produce antibodies specific to the antigens were separatedby the fluorescent antibody method and named ahMC5C12 (FERM BP-6070).The harvested clone cells were suspended in 90% bovine serum containing10% DMSO and kept in liquid nitrogen. The monoclonal antibodies,specific to mast cell surface antigens produced by the clones, wereharvested by growing the ahMC5C12 in the abdominal cavity of a nudemouse and purifying the antibodies.

INDUSTRIAL AVAILABILITY

It is possible to make clear the role of the mast cells in developmentof allergic diseases since the present invention identifies the aminoacid sequence of a mast cell surface antigen. Furthermore, since use ofan antibody to the mast cell surface antigen of the present inventionallows us to specifically remove or eliminate the mast cells, treatmentof allergic diseases becomes possible.

DEPOSITARY INSTITUTION

Hybridoma ahMC5C12 has been deposited with the following internationaldepositary institution, under the following accession number and depositdate.

Name: National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry

Address: 1-3, Higashi 1-home, Tsukuba city, Ibaraki pref., Japan

(Zip Code 305-0046)

Accession Number and Deposit Date:

(1) hybridoma ahMC5C12

FERM BP-6070: Aug. 21, 1997

1-3. (canceled)
 4. An antibody that specifically reacts against anisolated mast cell surface antigen comprising an amino acid sequencelisted in SEQ ID NO. 1.